Covalent binding of the benzamide RH-4032 to tubulin in suspension-cultured tobacco cells and its application in a cell-based competitive-binding assay.
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 [(14)C]RH-4032, and analysis of radiolabeled protein revealed a highly specific covalent attachment to beta-tubulin. Binding of [(3)H]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 [(3)H]RH-4032 was inhibited by the benzamides, pronamide and zarilamide, the N-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 [(3)H]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 [(3)H]RH-4032 was correlated with the ability to inhibit tobacco root elongation. The results suggest a common binding site on beta-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.
Young
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Rohm and Haas Company, Research Laboratories, Spring House, Pennsylvania 19477, USA. rsadhy@rohmhaas.com
Lewandowski
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D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism*; D001549:Benzamides / Q000378:metabolism*; D001667:Binding, Competitive; D002478:Cells, Cultured; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism*; D008938:Mitosis; D018517:Plant Roots / Q000254:growth & development / Q000378:metabolism; D010947:Plants, Toxic; D011869:Radioligand Assay; D008941:Spindle Apparatus / Q000378:metabolism; D014026:Nicotiana / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D014404:Tubulin / Q000378:metabolism*; D050257:Tubulin Modulators
D000590:Amino Acid Chloromethyl Ketones; D001549:Benzamides; C415979:RH 4032; D014404:Tubulin; D050257:Tubulin Modulators