Covalent Binding of the Benzamide RH-4032 to Tubulin in Suspension-Cultured Tobacco Cells and Its Application in a Cell-Based Competitive-Binding Assay

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Abstract
The benzamide, RH-4032, was found to be a potent antimicrotubule agent in tobacco (Nicotiana tabacum) cells. It strongly inhibited root growth and produced swollen club-shaped roots, an accumulation of cells in arrested metaphase, and loss of microtubules. RH-4032 inhibited the in vitro assembly of bovine tubulin into microtubules, with inhibition requiring a relatively long incubation period. Treatment of tobacco suspension-cultured cells or isolated bovine tubulin with [14C]RH-4032, and analysis of radiolabeled protein revealed a highly specific covalent attachment to β-tubulin. Binding of [3H]RH-4032 in tobacco suspension-cultured cells was shown to be saturable and to be influenced by pre-incubation of the cells with various antimicrotubule agents: Binding of [3H]RH-4032 was inhibited by the benzamides, pronamide and zarilamide, theN-phenylcarbamate, chlorpropham, and the microtubule-stabilizing drug, paclitaxel, whereas trifluralin and amiprophosmethyl were not inhibitory. A common characteristic of agents that cause microtubule disassembly was a slight enhancement of [3H]RH-4032 binding at low concentrations, which did not occur with the microtubule-stabilizing agent paclitaxel. For structural analogs of RH-4032 and various N-phenylcarbamates, it was shown that the ability to inhibit binding of [3H]RH-4032 was correlated with the ability to inhibit tobacco root elongation. The results suggest a common binding site on β-tubulin for RH-4032, pronamide, zarilamide, and chlorpropham, which is distinct from the binding site(s) for trifluralin and amiprophosmethyl. RH-4032 provides a unique approach to studying effects of antimicrotubule agents on plant cells by allowing competitive tubulin binding assays to be conducted in whole cells.