Differential in vitro action of S-12363, a new vinblastine derivative, and of its epimer on microtubule proteins

Wright, Michel; Garès, Michèle; Verdier-Pinard, Pascal; Moisand, A.; Berlion, Maryse; Legrand, Jean Jacques; Bizzari, Jean Pierre

doi:10.1007/bf00685819

Cited by 4 publications

(1 citation statement)

References 28 publications

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“…By contrast, polymerization induced by vinblastine occurs readily at 0°C, and the preformed polymer is cold-stable (18,20,24,33,34); we have confirmed this cold stability.…”

Section: Discussionsupporting

confidence: 69%

Localization of Critical Histidyl Residues Required for Vinblastine-induced Tubulin Polymerization and for Microtubule Assembly

Sadananda

Wolff

1998

Journal of Biological Chemistry

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Vinblastine-induced tubulin polymerization is electrostatically regulated and shows pH dependence with a pI ϳ7.0 suggesting the involvement of histidyl residues. Modification of histidyl residues of tubulin with diethylpyrocarbonate (DEPC) at a mole ratio of 0.74 (DEPC/ total His residues) for 3 min at 25°C completely inhibited vinblastine-induced polymerization with little effect on microtubule assembly. Under these conditions DEPC reacts only with histidyl residues. For complete inhibition two histidyl residues have to be modified. Demodification of the carboxyethyl histidyl derivatives by hydroxylamine led to nearly complete recovery of polymerization competence. Labeling with [ 14 C]DEPC localized both of these histidyl residues on ␤-tubulin at ␤227 and ␤264. Similarly, tubulin modification with DEPC for longer times (8 min) resulted in complete inhibition of microtubule assembly, at which time ϳ4 histidyl residues had been modified. This inhibition by DEPC was also reversed by hydroxylamine. The third histidyl residue was found on ␣-tubulin at ␣88. Thus, two charged histidyl residues are obligatorily involved in vinblastine-induced polymerization, whereas a different histidyl residue on a different tubulin monomer is involved in microtubule assembly.Tubulin can polymerize to rings, microtubules, sheets, bundles, and spirals depending on incubation conditions, drugs, etc. We have recently shown that vinblastine-induced tubulin polymerization into spiral structures is a two-step process with a critical concentration and a latent period for polymerization that is shortened by addition of polymer seeds. The process is similar to microtubule assembly in a number of respects, but unlike the latter, it is extremely sensitive to the presence of GTP and other oligoanions (1). This anion sensitivity resides primarily in the extreme C terminus of the ␤ monomer since removal of that portion of the monomer by subtilisin abolishes the anion inhibition (2). We proposed that the ␤-C terminus had to interact with a positively charged domain of tubulin to form vinblastine-induced spirals, a process that is interrupted and/or competed for by oligoanions. pH titration of the vinblastine-induced reaction revealed a cooperative inhibition of polymerization over the pH range of 6.5-7.3 with a pI near 7.0. This suggested that histidyl residue(s) might be part of the cationic domain required for spiral formation and the oligoanion promoted inhibition. Accordingly, we have studied the effects of modifications of critical His residues of tubulin by diethylpyrocarbonate (DEPC) 1 on vinblastine-induced spiral formation, and we have compared these to changes produced in microtubule assembly. The rat brain tubulin dimer contains 24 histidyl residues. Because the rate of histidyl reaction toward DEPC varies widely between and within proteins (3-7), such an approach would be useful only if the critical residues were also the most reactive ones. This proved to be the case, and two highly reactive histidyl residues were found to be required ...

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“…By contrast, polymerization induced by vinblastine occurs readily at 0°C, and the preformed polymer is cold-stable (18,20,24,33,34); we have confirmed this cold stability.…”

Section: Discussionsupporting

confidence: 69%