Cytochalasin a inhibits the invitro polymerization of brain tubulin and muscle actin

doi:10.1016/0006-291X(76)90346-6

Biochemical and Biophysical Research Communications

Volume 68, Issue 4, 23 February 1976, Pages 1362-1370

https://doi.org/10.1016/0006-291X(76)90346-6 Get rights and content

Abstract

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.

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Cited by (29)

Exploiting the cytoskeletal filaments of neoplastic cells to potentiate a novel therapeutic approach
2014, Biochimica et Biophysica Acta - Reviews on Cancer
Citation Excerpt :
Although cytochalasins are noted for their propensity to disrupt microfilaments, the unique α, β-unsaturated ketone moiety of cytochalasin A allows the congener to readily react with thiols [54,55]. As such, it has been shown that cytochalasin A reacts with critical thiol moieties on microtubules, with a binding site very similar to colchicine [56]. This interaction severely perturbs microtubules, thereby potentiating a novel mechanism by which the compound can damage malignant cells.
Although cytoskeletal-directed agents have been a mainstay in chemotherapeutic protocols due to their ability to readily interfere with the rapid mitotic progression of neoplastic cells, they are all microtubule-based drugs, and there has yet to be any microfilament- or intermediate filament-directed agents approved for clinical use. There are many inherent differences between the cytoskeletal networks of malignant and normal cells, providing an ideal target to attain preferential damage. Further, numerous microfilament-directed agents, and an intermediate filament-directed agent of particular interest (withaferin A) have demonstrated in vitro and in vivo efficacy, suggesting that cytoskeletal filaments may be exploited to supplement chemotherapeutic approaches currently used in the clinical setting. Therefore, this review is intended to expose academics and clinicians to the tremendous variety of cytoskeletal filament-directed agents that are currently available for further chemotherapeutic evaluation. The mechanisms by which microfilament directed- and intermediate filament-directed agents damage malignant cells are discussed in detail in order to establish how the drugs can be used in combination with each other, or with currently approved chemotherapeutic agents to generate a substantial synergistic attack, potentially establishing a new paradigm of chemotherapeutic agents.
Studies on the nocodazole-induced GTPase activity of tubulin
1996, Archives of Biochemistry and Biophysics
The tubulin dimer contains two guanine nucleotide binding sites, a nonexchangeable site occupied by GTP and an exchangeable site (E-site) occupied by GTP or GDP. Under the conditions used in this study the E-site GTP was hydrolyzed at a rate of 8 × 10⁻⁵s⁻¹at 37°C. This rate is stimulated four- to fivefold by nocodazole, an antimitotic drug. We studied the characteristics of this drug-stimulated reaction to learn more about the hydrolytic center of tubulin. The reaction, studied using single turnover kinetics, i.e., in the absence of added GTP, has a pH optimum of 6.6 to 7.2 and an activation energy of 88 kJ·mol⁻¹. It is first-order with respect to tubulin-GTP, indicating that hydrolysis is not dependent on an aggregation process. Divalent cations stimulate the hydrolysis three- to sixfold over the rate in the presence of EDTA. The reaction has a requirement for Na⁺that is not satisfied effectively by other monovalent cations. In contrast, Na⁺and K⁺are almost equally effective in the tubulin assembly reaction. Different purine nucleoside triphosphates can bind to the E-site and are hydrolyzed. GTP and ITP are hydrolyzed at equivalent rates and XTP and ATP are hydrolyzed at a rate about half as fast. Hydrolysis is inhibited by the reagents diethylpyrocarbonate andN-ethylmaleimide, although the GTPase activity is less sensitive than the assembly reaction. The reaction rate in D₂O is twice the rate in H₂O. This inverse isotope effect suggests the involvement of a sulfhydryl group in the rate-limiting step in the reaction.
Differential effects of anticytoskeletal compounds on the localization and chemical patterns of actin in germinating conidia of Neurospora crassa
1993, FEMS Microbiology Letters
Anti-actin drugs, cytochalasins A and B, inhibited both normal single, and benomyl-induced multiple, germ tube outgrowth from conidia of Neurospora crassa. Actin was cytochemically found to be concentrated in each of the benomyl-induced germ tube tips. No significant quantitative changes either in total actin or its isoforms were measured in the inhibitor-treated germlings. While intact microtubules are required for normal, monopolar axiation of the germ tube, they appear not to be necessary for benomyl-induced multipolar outgrowth which, in contrast, still requires intact actin microfilaments. Microfilaments and microtubules thus play complementary roles in the normal germination of conidia.
Tubulin sulfhydryl groups as probes and targets for antimitotic and antimicrotubule agents
1991, Pharmacology and Therapeutics
The sulfhydryl groups of tubulin are highly reactive entities. The reactivity of the sulfhydryl groups is sensitive to the presence of tubulin ligands, making these groups excellent probes for the interaction of tubulin with ligands. When tubulin is reacted with N,N′-ethylenebis-(iodoacetamide), two intrachain cross-links form in the β subunit. Formation of one of these cross-links is completely blocked by colchicine, podophyllotoxin, and nocodazole; formation of the other is blocked completely by maytansine, phomopsin A and GTP and partly by Vinca alkaloids. Different ligands also differ in their effect on the rate of alkylation of tubulin with iodo[¹⁴C]acetamide, with vinblastine and phomopsin A being strong inhibitors and maytansine having very little effect. Oxidation of certain key sulfhydryl groups can inhibit microtubule assembly. One of these sulfhydryl groups appears to be cys₂₃₉, but there are others not yet identified. Sulfhydryl-oxidizing agents also interfere with microtubule-mediated processes in vivo, raising the question of the existence of a physiological regulator of microtubule assembly. Potential physiological regulators have been examined to see if they can control microtubule assembly in vitro at their physiological concentrations. Of the ones that have been examined, thioredoxin and thioredoxin reductase are much better candidates for being physiological regulators than are either cystamine or glutathione.
Chemical modification of thiol group(s) in protein: Application to the study of anti-microtubular drugs binding
1987, Comparative Biochemistry and Physiology -- Part B: Biochemistry and
- 1.
  1. Different chemical procedures such as performic oxidation, carboxymethylation, carboxyethylation, aminoethylation, cyanylation, acylation, arylation etc. and addition of thiols to activated double bonds, titration of thiols with DTNB (Dithiobis-Nitro-Benzoate) and the reaction of thiols with organomercurials and the titration with p-chloro-mercuri-benzoate (PCMB) etc. are cited and discussed. Their chemical reactions are shown in the figures.
- 2.
  2. We describe in this paper that several chemicals interfere with microtubule assembly by combining with sulfhydryl residues. Reagents such as Cytochalasin-A and B, ethylacetylacrylate, FDNB (fluorodinitrobenzene), NEM (N-ethyl-maleimide), diamide, EBI (ethylene-bis-iodoacetamide, ethacrynic acid, methal ions, methylmercury, triethyllead ion and CDDP (cis-dichlorodiammine-platinium-II) are cited and their mechanisms are discussed.
Interaction of reduced glutathione with bovine brain tubulin
1985, Biochemical and Biophysical Research Communications
Incubation of phosphocellulose-purified tubulin with GSH at 30°C results in an inhibition of colchicine binding activity. GSSG has a protective effect against the GSH-induced loss of colchicine-binding. Incubation of tubulin with GSH at 30°C results in the formation of abnormal tubulin polymers which are insensitive to cold. Such aggregation is insensitive to antimicrotubular drugs. Aggregation is inhibited by GSSG but not by DTT or mercaptoethanol. GSH-induced aggregation is very sensitive to the ionic strength of the assembly medium; both the aggregation and colchicine binding inhibition induced by GSH are inhibited at higher ionic strength. These results indicate a very complex interaction of GSH with tubulin.

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Cytochalasin a inhibits the invitro polymerization of brain tubulin and muscle actin

Abstract

Endeavour

J. Bio. Chem

Nature

Nature

Science

J. Cell Biol

Cytochalasin a inhibits the $in vitro$ polymerization of brain tubulin and muscle actin