Sulfhydryl Groups Of Tubulin Research Articles

Biochemical characterization and molecular dynamic simulation of β-sitosterol as a tubulin-binding anticancer agent

Βeta-sitosterol (β-SITO), a phytosterol present in pomegranate, peanut, corn oil, almond, and avocado, has been recognized to offer health benefits and potential clinical uses. β-SITO is orally bioavailable and, as a constituent of edible natural products, is considered to have no undesired side effects. It has also been considered as a potent anticancer agent. However, the molecular mechanism of action of β-SITO as a tubulin-binding anticancer agent and its binding site on tubulin are poorly understood. Using a combination of biochemical analyses and molecular dynamic simulation, we investigated the molecular details of the binding interactions of β-SITO with tubulin. A polymer mass assay comparing the effects of β-SITO and of taxol and vinblastine on tubulin assembly showed that this phytosterol stabilized microtubule assembly in a manner similar to taxol. An 8-anilino-1-naphthalenesulfonic acid assay confirmed the direct interaction of β-SITO with tubulin. Although β-SITO did not show direct binding to the colchicine site on tubulin, it stabilized the colchicine binding. Interestingly, no sulfhydryl groups of tubulin were involved in the binding interaction of β-SITO with tubulin. Based on the results from the biochemical assays, we computationally modeled the binding of β-SITO with tubulin. Using molecular docking followed by molecular dynamic simulations, we found that β-SITO binds tubulin at a novel site (which we call the ‘SITO site’) adjacent to the colchicine and noscapine sites. Our data suggest that β-SITO is a potent anticancer compound that interferes with microtubule assembly dynamics by binding to a novel site on tubulin.

Methylmercury neurotoxicity: Role of oxidative stress

Methylmercury (MeHg) is a highly toxic compound that successfully traverses the blood brain barrier with deleterious effects to the central nervous system. Exposure is generally through the ingestion of contaminated fish. Two well-known disastrous exposures to methylmercury occurred in Japan in the 1950s and in Iraq in the 1970s. Acute methylmercury exposures such as these resulted in parasthesia, ataxia, and narrowing of the visual field. Exposures in utero resulted in microcephaly, cerebropalsy, seizures, and mental retardation. Since the developing fetus as well as the neonate are particularly vulnerable, the study of chronic low-dose exposures in utero which may result in developmental delays has become increasingly of interest. The known neurotoxic mechanisms are multifactorial. Methylmercury inhibits protein synthesis by post-transcriptionally interfering with chain elongation resulting from inhibition of aminoacyl-tRNA synthetase enzymes. Abnormalities in protein phosphorylation, critical to intercellular communication, were found both in vitro and in vivo. Methylmercury's high affinity for tubulin sulfhydryl groups was implicated in the disruption of microtubules. The resulting interference with tubulin polymerization can negatively affect cell proliferation and migration. Methylmercury altered neurotransmitter functions by stimulating spontaneous transmitter release and inhibiting glutamate uptake resulting in excitotoxicity. Methylmercury disrupted Ca2+ homeostasis by increasing intracellular concentrations of this ion. Methylmercury may overwhelm the cell's natural protective mechanisms provided by glutathione, metallothioneins, and heat shock protein, thereby producing the generation of reactive oxygen species (ROS). The resulting oxidative stress produced damage to cellular membranes through lipid peroxidation as well as damage to a variety of vital cellular macromolecules. The observed protective effect of antioxidants supports the critical role of oxidative stress in neurotoxicity. Though much has already been discerned regarding the various mechanisms involved in methylmercury-induced neurotoxicity, the interplay between these modes and whether or not they occur sequentially or synchronously has yet to be determined. Attention in this review is focused on oxidative stress since recent studies suggests that oxidative stress may be the initiating event from which other modes of cellular damage originate.

Allicin inhibits cell polarization, migration and division via its direct effect on microtubules

Allicin (diallyl thiosulfinate) is a major biologically active component of garlic that is known to inhibit cell proliferation and induce apoptosis. The effects of allicin are attributed to its ability to react with thiol groups. However, the mechanism underlying the cytostatic activity of allicin, as well as the identity of the relevant subcellular targets, are not known. In the present study, we found that the effects of allicin on cell polarization, migration, and mitosis are similar to the effects of microtubule-depolymerizing drugs such as nocodazole. Moreover, treatment of cultured fibroblasts with micromolar doses of allicin results in microtubule depolymerization in cells within minutes of its application, without disrupting the actin cytoskeleton or inducing direct cytotoxic effects. Furthermore, allicin blocks the polymerization of pure tubulin in vitro in a concentration-dependent manner, suggesting that it acts directly on tubulin dimers. Sulfhydryl (SH)-reducing reagents such as 2-mercaptoethanol and dithiothreitol abolish the effect of allicin on microtubule polymerization. Thus, allicin is a potent microtubule-disrupting reagent interfering with tubulin polymerization by reaction with tubulin SH groups.

Cytoskeleton/Endoplasmic Reticulum Collapse Induced by Prostaglandin J2 Parallels Centrosomal Deposition of Ubiquitinated Protein Aggregates

Many neurodegenerative disorders, such as Parkinson disease, exhibit inclusion bodies containing ubiquitinated proteins. The mechanisms implicated in this aberrant protein deposition remain elusive. In these disorders signs of inflammation are also apparent in the affected central nervous system areas. We show that prostaglandin J2 (PGJ2), an endogenous product of inflammation, disrupts the cytoskeleton in neuronal cells. Furthermore, PGJ2 perturbed microtubule polymerization in vitro and decreased the number of free sulfhydryl groups on tubulin cysteines. A direct effect of PGJ2 on actin was not apparent, although actin filaments were altered in cells treated with PGJ2. This cyclopentenone prostaglandin triggered endoplasmic reticulum (ER) collapse and the redistribution of ER proteins, such as calnexin and catechol-O-methyltransferase, into a large centrosomal aggregate containing ubiquitinated proteins and alpha-synuclein. The PGJ2-dependent cytoskeletal rearrangement paralleled the development of the large centrosomal aggregate. Both of these events were replicated by treating cells with colchicine, which disrupts the microtubule/ER network, but not with brefeldin A, which impairs ER/Golgi transport. PGJ2 also perturbed 26 S proteasome assembly and activity, which preceded the accumulation of ubiquitinated proteins as detergent/salt-insoluble aggregates. Our data support a mechanism by which, upon PGJ2 treatment, cytoskeleton/ER collapse coincides with the relocation of ER proteins, other potentially neighboring proteins, and ubiquitinated proteins into centrosomal aggregates. Development of these large perinuclear aggregates is associated with disruption of the microtubule/ER network. This aberrant protein deposition, triggered by a product of inflammation, may be common to other compounds that disrupt microtubules and induce protein aggregation, such as MPP+ and rotenone, found to be associated with neurodegeneration.

Complete activation of porcine oocytes induced by the sulfhydryl reagent, thimerosal.

Thimerosal (200 microM) triggered Ca2+ oscillations in 56 of 56 mature porcine oocytes. The Ca2+ oscillations were blocked by the sulfhydryl-reducing agent dithiothreitol (DTT), thus supporting the hypothesis that thimerosal acts by oxidizing critical sulfhydryl groups on intracellular Ca2+-release proteins. Thimerosal treatment alone arrested the oocytes in metaphase, probably by oxidizing tubulin sulfhydryl groups and thus destroying the spindle. However, a 10-min exposure to 200 microM thimerosal followed by a 30-min incubation in 8 mM DTT induced complete activation, as 73.8% of the oocytes formed pronuclei. The second polar body was visible in 73.3% (55 of 75) of the activated oocytes. Combined thimerosal/DTT treatment of the oocytes also induced cortical granule exocytosis, as revealed by confocal microscopy, and the subsequent hardening of the zona pellucida. After activation, some oocytes were incubated in vitro, or in vivo in a ligated porcine oviduct, for 6 days. When cultured in vitro, 42.0% (37 of 88) of the oocytes developed to the compact morula or blastocyst stage; the average number of inner cell mass (ICM) and trophectoderm (TE) nuclei in the blastocysts was 8.6 +/- 0.7 and 20.1 +/- 1.3, respectively. Culture in a ligated oviduct resulted in 42.9% development to the compact morula or blastocyst stage, with the blastocysts having a mean number of 12.5 +/- 1.0 ICM and 63.6 +/- 9.2 TE nuclei.

Interaction of three sponge‐derived macrocyclic lactone polyethers (spongistatin 3, halistatins 1 and 2) with tubulin

AbstractMacrocyclic lactone polyethers of marine origin include a series of compounds which bind to tubulin, the subunit protein of microtubules, and inhibit microtubule assembly and vinblastine binding to tubulin. We have previously studied two of these compounds: halichondrin B and the very similar homohalichondrin B. We have found that halichondrin B enhances the time‐dependent exposure of hydrophobic areas on the tubulin molecule without affecting the exposure of the sulfhydryl groups, while homohalichondrin B has no effect on exposure of hydrophobic areas and slightly suppresses exposure of sulfhydryl groups. The spongistatins (isolated from the marine sponge Spongia sp.) resemble the halichondrins, except for having a smaller ring; spongistatin 1 inhibits vinblastine binding to tubulin and blocks microtubule assembly. Here, we have examined the effect of the closely related spongistatin 3 on the exposure of tubulin sulfhydryl groups and hydrophobic areas. We have found that spongistatin 3 inhibits formation of the same intrachain cross‐link in tubulin as is inhibited by vinblastine. Unlike vinblastine, however, spongistatin 3 has no effect on the exposure of either sulfhydryl groups or hydrophobic areas on the tubulin molecule. In short, spongistatin 3 resembles maytansine in its effects on tubulin more than it does either halichondrin B or homohalichondrin B. We also examined the interaction of tubulin with halistatins 1 and 2, which are close structural analogues of halichondrin B and homohalichondrin B, respectively. Halistatins 1 and 2 are isolated, respectively, from the marine sponges Phakellia carteri and Axinella carteri. We find that both halistatins resemble halichondrin B in that they have no effect on the exposure of sulfhydryl groups. Our results suggest that very small structural changes in these compounds can significantly alter the pattern of their effects on the tubulin molecule. © 1995 Wiley‐Liss, Inc.

Interaction of ethacrynic acid with bovine brain tubulin

Ethacrynic acid is a diuretic agent that reacts with sulfhydryl groups in proteins, and which shows promise of effectiveness in the treatment of glaucoma. Ethacrynic acid is a known inhibitor of microtubule assembly in vitro (Xu et al., Arch Biochem Biophys296: 462-67, 1992). We have used N,N′-ethylenebis (iodoacetamide) (EBI) as a probe to examine the sulfhydryl groups of tubulin; EBI can form two intra-chain cross-links in β-tubulin. One of these, β∗, connects Cys239 with Cys354; the other, βs, joins Cys12 with either Cys201 or Cys211 (Little and Ludueña, EMBO J4: 51–56, 1985; Biochim Biophys Acta912: 28–33, 1987). Formation of β∗ inhibits microtubule assembly in vitro, consistent with the hypothesis that Cys239 has an assembly-critical sulfhydryl (Bai et al., Biochemistry28: 5606–5612, 1989). We have examined the interaction of ethacrynic acid with the sulfhydryl groups of bovine brain tubulin. We found that 130 μM ethacrynic acid gave half-maximal inhibition of assembly, but had no effect on the formation of the β∗ cross-link by EBI. Ethacrynic acid, however, did inhibit substantially formation of the βs cross-link at this concentration and half-maximally inhibited it at approximately 185 μM. Half-maximal inhibition of the alkylation of tubulin sulfhydryls by iodo [14C]acetamide was obtained at an ethacrynic acid concentration in the range of 190–325 μM. These results indicate that ethacrynic acid can inhibit microtubule assembly by reacting with sulfhydryl groups other than those of Cys239 and Cys354 and suggest that other sulfhydryl groups in tubulin could be assembly-critical. These results also raise the possibility that these other assembly-critical sulfhydryls may be those of Cys12, Cys201 or Cys211.

Interaction of halichondrin B and homohalichondrin B with bovine brain tubulin

Halichondrin B is a polyether macrolide of marine origin which binds to tubulin and inhibits microtubule assembly in vitro and in vivo. As is the case with phomopsin A and dolastatin 10, halichondrin B is a non-competitive inhibitor of vinblastine binding to tubulin. Analogous to maytansine, which by contrast is a competitive inhibitor of vinblastine binding, halichondrin B has no effect on colchicine binding, which is greatly stabilized by phomopsin A and dolastatin 10, but not by maytansine. We have previously developed assays which allow sensitive discrimination among the interactions of various ligands with tubulin, and examined the effects of ligands on the reactivity of tubulin sulfhydryl groups and the exposure of hydrophobic areas on the surface of the tubulin molecule. To classify the nature of the interaction between halichondrin B and tubulin, in this study we examined the effects of halichondrin B and its closely related analogue, homohalichondrin B, by these assays. We found that: (1) halichondrin B and homohalichondrin B both inhibited formation of an intra-chain cross-link between two sulfhydryl groups in β-tubulin, as do phomopsin A, dolastatin 10, maytansine, and vinblastine; (2) halichondrin B resembles maytansine in that it had no effect on alkylation of tubulin sulfhydryl groups by iodoacetamide, unlike phomopsin A, dolastatin 10 and vinblastine, all of which inhibit alkylation; (3) halichondrin B differs from other anti-mitotic drugs in that it enhanced exposure of hydrophobic areas on tubulin; (4) homohalichondrin B, like maytansine and in contrast to phomopsin A, dolastatin 10 and vinblastine, had no effect on exposure of hydrophobic areas; and (5) homohalichondrin B, contrary to maytansine, inhibited alkylation of tubulin sulfhydryl groups in the presence of GTP and MgCl 2. In their interactions with the tubulin molecule, halichondrin B and homohalichondrin B appear to have unique conformational effects which differ from those of other drugs and also from the effects of each other as well.

Selenium: Inhibition of microtubule formation and interaction with tubulin

We have studied the interaction of Na 2SeO 3 with microtubule proteins and tubulin. This selenium compound inhibits the polymerization of MTP (half-inhibition occurred for Na 2SeO 3 10 μM), and to a lesser that of tubulin. This effect of selenite is related to the formation of disulfide bridges between tubulin sulfhydryl groups, inducing a conformational change of the protein. This is corroborated by the modified binding of colchicine and vinblastine in presence of selenium. The selenite inhibitory concentrations are similar to the toxic blood levels of selenium (40 μM).

Cisplatin toxicity: effects on phagocyte functions

The in vitro effects of cisplatin on human polymorphonuclear functions were studied in order to establish if the drug is able, during chemotherapy, to induce an immune depression of host response and an increased susceptibility to infections. All the phagocytic functions tested were negatively influenced, but only chemotaxis was really depressed by cisplatin used in therapeutic concentrations (from 10 −5 to 10 −6 M). This finding was confirmed in chemotactic tests performed on leukocytes elicited from cancer patients after cisplatin intravenous administration. The binding to the tubulin sulfhydryl groups and the inhibition of microtubule polymerization have been proposed to explain how the drug affects cytoskeleton activities.

The effects of the anilinonaphthalenesulfonates on the alkylation of tubulin: correlation between the appearance of sulfhydryl groups and apolar binding sites

We have previously found that the sulfhydryl groups of tubulin are sensitive reporters of the effects of ligands on the tubulin molecule. In this study, we examined the effects of three anilinonaphthalenesulfonates on the interaction of tubulin with iodo[ 14C]acetamide and N, N′-ethylenebis(iodoacetamied). We found that 1,8-anilinonaphthalensulfonate (1,8-ANS) and 2,6-anilinonaphthalenesulfonate (2,6-ANS) had no effect on the ractiion with iodo[ 14C]acetamide. In contrast, bis(1,8-anilinonaphthalenesulfonate) (BisANS), an inhibitor of microtubule assembly, had a complex effect. Low concentrations of BisANS, where presumably only the high-affinity binding site was saturated, had little or no effect on alkylation. Higher concentrations of BisANS cause a strong enhancement of alkylation. None of these compounds had any effect on the reaction with N, N′-ethylenebis(iodoacetamide). Our results suggest that the binding of BisANs, 2,6-ANS and 1,8-ANS to tubulin is complex and very different from that of the other anti-tubulin drugs. The correlatiion between the effects of drugs on alkylation of tubulin and the binding of BisANS is consistent with a model whereby the alkylatable sulfhydryls are located in apolar regions of the tubulin molecule.

Bis(8-anilinonaphthalene-1-sulfonate) as a probe for tubulin decay

The fluorescent apolar probe bis(8-anilinonaphthalene-1-sulfonate) (Bis-ANS) has been used to detect structural correlates of the well-known but poorly understood decay of tubulin function, by which tubulin loses its ability to polymerize and bind drugs in a complex, time-dependent way. The present results indicate that the decay of tubulin is accompanied by the appearance of hydrophobic areas, which bind a total of six Bis-ANS molecules with a dissociation constant of 19 microM. This binding seems to be a result of localized structural changes that are taking place in the tubulin molecule and can be used as a probe for these changes. In particular, circular dichroism measurements revealed no significant changes in the average secondary structure of the protein during the time required for complete binding of the Bis-ANS molecules. Preincubation of tubulin with the antimitotic drugs colchicine, podophyllotoxin, and vinblastine slows the rate of appearance of the hydrophobic region. Vinblastine has the maximal effect followed by colchicine and podophyllotoxin. In contrast, preincubation with maytansine has no effect. In addition, lowering the temperature decreases the rate of appearance of this region. These results correlate with the effect of drugs on the alkylation of tubulin sulfhydryl groups by iodoacetamide [Luduena, R.F., & Roach, M.C. (1981) Biochemistry 20, 4444-4450] and with the ability of inhibitors of microtubule assembly to permit the polymerization of tubulin into nonmicrotubule structures.

N,N-Bis(α-iodoacetyl)-2,2′-dithiobis(ethylamine), a reversible crosslinking reagent for protein sulfhydryl groups

This paper describes the synthesis and analysis of N,N′-bis(α-iodoacetyl)-2-2′-dithiobis(ethylamine) (BIDBE), a bifunctional reagent with the structure ICH 2CONH-(CH 2) 2SS(CH 2) 2NHCOCH 2I. BIDBE is shown to react with the sulfhydryl groups of tubulin and to generate a cleavable crosslink between aldolase subunits. BIDBE may be the only reagent hitherto described which can generate a cleavable crosslink between nonadjacent sulfhydryl groups in proteins.

Contrasting effects of maytansine and vinblastine on the alkylation of tubulin sulfhydryls

The ansa macrolide maytansine is a competitive inhibitor of vinblastine for binding to tubulin. Both drugs are potent inhibitors of microtubule assembly in vitro but maytansine, unlike vinblastine, is unable to induce tubulin aggregation or to stabilize colchicine binding. In this study, the effects of maytansine and vinblastine on the accessibility of tubulin's sulfhydryl groups were compared. It was found that 10 μ m vinblastine inhibited the reaction of bovine brain tubulin with [ 14C]iodoacetamide by 45%. In contrast, maytansine, even up to 100 μ m, had no effect on the reaction. However, when the two drugs were tested in combination, maytansine was a potent inhibitor of vinblastine's effect, consistent with the two drugs competing for the same or overlapping sites, but suggesting that the nature of the binding was different. In contrast, maytansine did not affect the suppression of alkylation induced by colchicine and podophylotoxin, consistent with these drugs binding to different sites. Maytansine and vinblastine were each able to increase the formation of β ∗ by the bifunctional reagent, N,N′-ethylenebis-(iodoacetamide); β ∗ is the designation for an electrophoretically faster migrating form of β-tubulin which apparently contains an intrachain crosslink. Thus, in at least the portion of the tubulin molecule involved in β ∗ formation, the two drugs have similar effects. Since maytansine does not appear to suppress any competing alkylation reactions, it is possible that the enhancement of β ∗ formation represents a genuine conformational effect. Since the sulfhydryl groups of tubulin may be involved in regulating microtubule assembly, it is likely that maytansine and vinblastine differ in the manner in which they inhibit microtubule assembly.

Interaction of tubulin with drugs and alkylating agents. 2. Effects of colchicine, podophyllotoxin, and vinblastine on the alkylation of tubulin.

The antimitotic drugs colchicine, podophyllotoxin, and vinblastine are known to be potent inhibitors of microtubule polymerization, but little is known about how they affect the chemical properties of the tubulin molecule. In the preceding paper [Ludueña, R. F., & Roach, M. C. (1981) Bio-chemistry (preceding paper in this issue)], we have shown that the alkylating agent iodo[14C]acetamide reacts specifically with the sulfhydryl groups of tubulin and that its bifunctional analogue, N,N'-ethylenebis(iodoacetamide) (EBI), reacts with native tubulin to convert beta-tubulin into a form, designated beta*, which appears to represent an intrachain cross-linked form of beta. In this paper, we have incubated tubulin with the drugs prior to alkylation and measured their effects on the alkylation reactions. We have found that at 100 microM concentrations, podophyllotoxin, colchicine, and vinblastine inhibited the reaction of tubulin with iodo[14C]acetamide by 19-32%, 33-47%, and 62-72%, respectively; each drug was half-maximally effective at 3-5 microM, indicating that the suppressive effects of the drugs were mediated by their high-affinity binding sites. Similarly, beta* formation induced by EBI was suppressed by 92-94% in the presence of either colchicine or podophyllotoxin In contrast, vinblastine enhanced beta* formation by 40%. Alkylation with longer chain analogues of EBI revealed no evidence that the reactive sulfhydryls were being pushed apart by the drugs. These results indicate that each of the drugs has potent effects on the accessibility of the sulfhydryl groups of tubulin and that the effects of vinblastine are very different from those of either colchicine or podophyllotoxin.

Interaction of tubulin with drugs and alkylating agents. 1. Alkylation of tubulin by iodo[14C]acetamide and N,N'-ethylenebis(iodoacetamide).

The sulfhydryl groups of tubulin are reported to play a role in regulating microtubule assembly and colchicine binding to tubulin. The alkylating agents iodo[14C]acetamide and its bifunctional analogue N,N'-ethylenebis(iodoacetamide) are used as probes for the sulfhydryl groups of tubulin. In the presence of 8 M urea, alpha- and beta-tubulin have 10-11 and 8 alkylatable sulfhydryls, respectively, and one of the high molecular weight proteins (HMW 2) has 5 sulfhydryls/Mr 271 000. In the absence of urea, the rates of alkylation of alpha- and beta-tubulin are significantly lower but that of HMW 2 is unaffected. The sulfhydryls of tubulin are masked in intact microtubules. N,N'-Ethylenebis(iodoacetamide) reacts with free tubulin to generate a band, designated beta, which migrates ahead of beta on polyacrylamide gels. beta appears to represent a form of beta-tubulin containing at least one intrachain cross-link between sulfhydryl groups. Formation of beta* is inhibited in intact microtubules and is abolished if tubulin is denatured by 8 M urea, 1% sodium dodecyl sulfate, or boiling. N,N'-Ethylenebis(iodoacetamide) may thus be used as a probe for the native conformation of free tubulin.

Lactoperoxidase-tubulin interactions.

Self-iodinated lactoperoxidase co-polymerizes with brain microtubules to constant specific activity and a stoichiometry of 0.2 to 0.3 lactoperoxidase molecule/tubulin heterodimer polymerized in the presence of 4 M glycerol. By contrast, iodinated tubulin loses its competence to polymerize. The lactoperoxidase-microtubule association is salt- and temperature-sensitive, shows considerable specificity, is saturable, and is reversible. Lactoperoxidase does not displace the microtubule-associated proteins from microtubules, does not promote polymerization, and binds to preformed microtubules. Self-iodinated lactoperoxidase also binds to tubulin oligomers at 0 degrees C and in the presence of CaCl2. The stoichiometry for this interaction is 0.6 to 0.8 molecules of enzyme/dimer. Lactoperoxidase forms a complex with soluble brain tubulin prepared by two cycles of polymerization and depolymerization or by phosphocellulose chromatography. The interaction was studied by sucrose gradient analysis, gel filtration, and spectral analysis based on the finding that tubulin binding to lactoperoxidase leads to a red shift in the Soret spectrum, yielding a difference spectrum with a minimum of 410 nm and maximum at 430 nm. This interaction involves one or more sulfhydryl groups of tubulin. Complex formation is relatively slow, is retarded by 0.6 M NaCl, and is accelerated by diiodotyrosine. By all three methods of analysis, the stoichiometry approaches a value of 2 lactoperoxidase molecules/tubulin dimer. There is a single class of binding sites in pig, beef, or rat tubulin with an apparent overall affinity constant of approximately 2.0 x 10(6) M-1. The molecular weight of the complex by sucrose gradient or gel filtration is approximately 140,000 i.e. half of the expected value for a 2:1 adduct. Since both alpha and beta subunits are present in the complex, we propose that the complex consists of a mixture of equal parts of presumably native alpha-tubulin-lactoperoxidase and beta-tubulin-lactoperoxidase.

Inhibition of tubulin assembly by ethylacetylacrylate, a sulfhydryl reagent and potential analog of cytochalasin A

Ethyl acetylacrylate reacts rapidly with 2-mercaptoethanol and the sulfhydryl groups of tubulin. The structure of ethyl acetylacrylate resembles that of the reactive portion of cytochalasin A and potentially can serve as an analog of this antibiotic.

Tubulin sulfhydryl groups and polymerization in vitro: Effects of di- and trivalent cations

The cations, Hg 2+, Cu 2+, Cd 2+, Ni 2+, Mn 2+, Co 2+, Zn 2+, Cr 3+ and Al 3+ have been investigated with respect to their effect on the polymerization and on the amount of free sulfhydryl groups of a brain tubulin preparation. Tubulin polymerization was completely inhibited by Hg 2+, Cu 2+, Cd 2+ and Cr 3+ at 10 −3 M but only slightly by Ni 2+ and Al 3+. In the presence of 10 −4 M Ni 2+ a stimulatory effect was observed. Co 2+ at 10 −3 M caused formation of sheet polymers of tubulin. The sheets were stable against colchicine and Ca 2+ depolymerization. Mn 2+ did not affect the polymerization. The amount of free sulfhydryl groups was markedly decreased by Hg 2+, Cu 2+ and Cd 2+ at 10 −3 M but less by Co 2+, Mn 2+ and Zn 2+. In the presence of Ni 2+, Cr 3+ and Al 3+ at the same concentration no effects were observed. The results indicate that some but not all free sulfhydryl groups of tubulin are directly involved in the polymerization process, and also that other metal binding groups could be of importance.

Cytochalasin a inhibits the [formula omitted] polymerization of brain tubulin and muscle actin

Cytochalasin A (CA) inhibits the self-assembly of beef brain tubulin. The concentrations necessary to cause the inhibition are only slightly higher than the tubulin concentration. Cytochalasin B (CB) at identical and higher concentrations has no noticeable effect. Cytochalasin A also inhibits colchicine binding activity suggesting that it denatures the tubulin molecule. The results indicate that the reaction of CA with the sulfhydryl groups of tubulin is responsible for its action. CA also prevents the conversion of G-actin to F-actin, probably via a similar mechanism.